Project Summary Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that is problematic for individuals who are immunocompromised. Importantly, P. aeruginosa is responsible for pathology associated with chronic colonization of individuals with cystic fibrosis. The organism is well adapted to health care settings by being able to survive on minimal nutrients, form microbial communities on surfaces and express intrinsic resistance to antibiotics and disinfectants. P. aeruginosa has a large repertoire of destructive enzymes and toxins that aid bacterial replication by neutralizing innate immune cells in human hosts. The mechanisms of action include interference with or inhibition of signal transduction, host protein synthesis, cytoskeletal function or membrane dynamics. Our studies focus on the cytotoxic enzymes or effectors injected by the type III secretion system of P. aeruginosa. These enzymes, ExoS and ExoU, demonstrate broad substrate specificity and can recognize both prokaryotic and eukaryotic targets. The property of broad substrate specificity requires that additional control elements must be in place to keep the effector inactive before delivery to the appropriate host environment. The type III effector, ExoU, a potent phospholipase, is used as a model system to study the dynamic changes that mediate activation of enzyme activity in host cells. We identified ubiquitin (Ub), which is synthesized only by eukaryotes, as the activator required for ExoU- mediated phospholipase activity in host cells. The long-term objectives of this research are to mechanistically describe Ub and substrate induced conformational changes that occur to activate ExoU. We have used molecular modeling, continuous wave and double electron electron resonance spectroscopy to build an initial model of ExoU activation. The model makes specific predictions and provides testable hypotheses that will be challenged in iterative biochemical and biophysical analyses and model refinement with novel mono- and diUb probes. Ub-mediated activation is postulated to serve as a specific, therapeutic target, not limited to ExoU. This property extends to ExoU-orthologs that are encoded in the genomes of a variety of Gram negative human pathogens and opportunists. Overall, these studies aim to contribute a structure-function basis for the rational design of inhibitors that may be applicable for treatment of infections caused by resistant, problematic organisms.